Microparticle sorting method and microchip for sorting microparticles

ABSTRACT

There is provided a microparticle sorting method including a procedure of collecting a microparticle in a fluid that flows through a main channel in a branch channel that is in communication with the main channel by generating a negative pressure in the branch channel. In the procedure, a flow of a fluid is formed that flows toward a side of the main channel from a side of the branch channel at a communication opening between the main channel and the branch channel.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a divisional application of U.S. patentapplication Ser. No. 13/963,188, filed Aug. 9, 2013, which claims thepriority from prior Japanese Priority Patent Application JP 2012-180317filed in the Japan Patent Office on Aug. 16, 2012, the entire content ofwhich is hereby incorporated by reference.

BACKGROUND

The present technology relates to a microparticle sorting method. Morespecifically, the present technology relates to a microparticle sortingmethod that separates and recovers only target microparticles from themicroparticles that are flowing along a channel.

A microparticle sorting apparatus that forms a microparticle-containingsheath flow in a channel, detects fluorescence and scattered lightemitted from the microparticles by irradiating light on themicroparticles in the sheath flow, and separates and recovers amicroparticle group (population) that exhibits a predetermined opticalcharacteristic is known. For example, in a flow cytometer, a specifictype of cell only is separated and recovered by labeling a plurality oftypes of cell included in a sample with a fluorescent dye and opticallyidentifying the fluorescent dye labeled on each cell.

In JP 2009-100698A and JP 2005-538727T, microchip-type microparticlesorting apparatuses are disclosed that perform analysis by forming asheath flow in a channel formed on a microchip that is made fromplastic, glass or the like.

The microparticle sorting apparatus disclosed in JP 2009-100698Acontrols the feeding direction of the sheath flow at a branching portionbetween an introduction channel in which the sheath flow is formed and abranch channel in communication with the introduction channel bygenerating an air bubble based on laser irradiation at the branchingportion. According to this microparticle sorting apparatus, controllingthe feeding direction of the sheath flow at the branching portion withan air bubble enables just the target microparticles to be collectedinto the branch channel from the introduction channel and sorted.

Further, the microfluidic system disclosed in JP 2005-538727T sortstarget microparticles by using an actuator to control the feedingdirection of a sheath flow at a channel branching portion. In thismicrofluidic system, the actuator changes the feeding direction of thesheath flow by pressing against a chamber that is connected to abranching portion between an introduction channel in which the sheathflow is formed and a branch channel in communication with theintroduction channel to push out fluid in the chamber.

SUMMARY

For microchip-type microparticle sorting apparatuses, in order tofurther increase the speed and accuracy of analysis, there is a demandfor a technology for rapidly and stably extracting only targetmicroparticles from a sheath flow that is flowing through a channel.

According to an embodiment of the present technology, there is provideda microparticle sorting technology that can rapidly and stably extractonly target microparticles from a sheath flow that is flowing through achannel.

According to an embodiment of the present technology, there is provideda microparticle sorting method including a procedure of collecting amicroparticle in a fluid that flows through a main channel in a branchchannel that is in communication with the main channel by generating anegative pressure in the branch channel. In the procedure, a flow of afluid is formed that flows toward a side of the main channel from a sideof the branch channel at a communication opening between the mainchannel and the branch channel. The flow may be formed by introducingthe fluid into the branch channel from an introduction openingpositioned near the communication opening in the branch channel. Thefluid introduced from the introduction opening into the branch channelis split into a counter flow that flows toward the communication openingand a forward flow that flows in the opposite direction.

In this microparticle sorting method, by maintaining the flow of thefluid formed in the communication opening that flows toward the mainchannel side from the branch channel side before and after theabove-described steps, the fluid in the main channel can be preventedfrom unnecessarily entering the branch channel during the period that anegative pressure is not being generated in the branch channel.

According to the microparticle sorting method of the present technology,in the procedure, a flow rate of the fluid that is sucked into thebranch channel from the main channel due to negative pressure may begreater than a flow rate of the fluid introduced into the branch channelfrom the introduction opening and fed toward the communication opening.The microparticle in the main channel may be hereby collected from thecommunication opening to a position that is past the introductionopening of the branch channel.

According to the microparticle sorting method of the present technology,in the procedure, the negative pressure may be generated by an actuatorapplying a force that deforms an inner space of the branch channel tocause a volume of the inner space to increase

A change in the negative pressure may have a pulse waveform, a stepwaveform, or an undershoot-step waveform.

According to an embodiment of the present technology, there is provideda microchip for sorting microparticles, including a sample fluidintroduction opening into which a sample fluid including a microparticleis introduced, a sample fluid channel through which the sample fluidintroduced from the sample fluid introduction opening flows, a sheathfluid introduction opening into which a sheath fluid is introduced, afirst sheath fluid channel through which the sheath fluid introducedfrom the sheath fluid introduction opening flows, a main channel wherethe sample fluid channel and the first sheath fluid channel merge, abranch channel that is in communication with the main channel, and asecond sheath fluid channel that connects the sheath fluid introductionopening and a sheath fluid discharge opening that is positioned near acommunication opening to the main channel in the branch channel, andthat feeds the sheath fluid introduced from the sheath fluidintroduction opening into the branch channel from the sheath fluiddischarge opening. According to the microchip for sorting microparticlesof the present technology, the second sheath fluid channel may not be incommunication with the sample fluid channel, the first sheath fluidchannel, or the main channel. An actuator for applying a displacement ona contact surface may be arranged in contact with a positioncorresponding to the branch channel on a surface. A pressure chamber forproducing a change in volume due to the displacement may be configuredin the branch channel. The communication opening, the sheath fluiddischarge opening, and the pressure chamber may be arranged in thebranch channel in order of mention. The microchip for sortingmicroparticles may further include the two first sheath fluid channels.The sheath fluid introduction opening may be provided at a symmetricalcenter of the two first sheath fluid channels. An end on an oppositeside to the communication opening of the branch channel may be an openend.

In an embodiment of the present technology, the term “microparticle” hasa broad meaning that includes biologically-relevant microparticles suchas cells, microbes, ribosomes and the like, as well as syntheticparticles such as latex particles, gel particles, industrial particlesand the like.

Examples of biologically-relevant microparticles include thechromosomes, liposomes, mitochondria, organelles (cell organelles) thatform various cells. Examples of cells include animal cells(hematopoietic cells etc.) and plant cells. Examples of microbes includebacteria such as E. coli, viruses such as tobacco mosaic virus, fungisuch as yeast and the like. Further example of biologically-relevantmicroparticles includes nucleic acids, proteins, complexes of these andthe like. Examples of industrial particles include organic or inorganicpolymer materials, metals and the like. Examples of organic polymermaterials include polystyrene, styrene-divinyl benzene, poly methylmethacrylate and the like. Examples of inorganic polymer materialsinclude glass, silica, magnetic materials and the like. Examples ofmetals include metal colloids, aluminum and the like. Although the shapeof these microparticles is usually spherical, the microparticles mayalso have a non-spherical shape. Further, the size and mass of thesemicroparticles is not especially limited.

According to the embodiments of the present technology described above,a microparticle sorting technology is provided that can rapidly andstably extract only target microparticles from a sheath flow that isflowing through a channel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a configuration of a microparticlesorting apparatus A according to a first embodiment of the presenttechnology;

FIG. 2 is a diagram illustrating a configuration of a microchip 1 a thatis mounted on a microparticle sorting apparatus A;

FIG. 3 is a diagram illustrating a configuration of the microchip 1 a;

FIG. 4 is a diagram illustrating a configuration of the microchip 1 a;

FIGS. 5A, 5B and 5C are diagrams illustrating a configuration of abranching portion between a main channel 15 and a sorting channel 16 ofthe microchip 1 a;

FIG. 6 is a diagram illustrating a configuration of a sheath fluid inlet13 side end of a sheath fluid bypass channel 18 of the microchip 1 a;

FIG. 7 is a diagram illustrating a configuration of a discharge opening181 side end of the sheath fluid bypass channel 18 of the microchip 1 a;

FIGS. 8A, 8B, 8C, 8D, 8E and 8F are diagrams illustrating a sortingoperation in the microparticle sorting apparatus A;

FIGS. 9A and 9B are diagrams illustrating functions of a pressurechamber 161 in the microchip 1 a;

FIG. 10 is a diagram illustrating a configuration of a modified exampleof the microchip 1 a;

FIG. 11 is a diagram illustrating a flow of a sample fluid and a sheathfluid that may be produced at a branching portion between the mainchannel 15 and the sorting channel 16;

FIGS. 12A and 12B are diagrams illustrating a flow of the sheath fluidintroduced from the discharge opening 181 of the sorting channel 16;

FIGS. 13A and 13B are diagrams illustrating a position where a targetparticle is drawn in during a sorting operation;

FIGS. 14A, 14B and 14C are diagrams illustrating waveforms of thevoltage applied on an actuator 31 from a drive unit 23; and

FIG. 15 is a diagram illustrating a configuration of a modified exampleof the microchip 1 a.

DETAILED DESCRIPTION OF THE EMBODIMENT(S)

Hereinafter, preferred embodiments of the present disclosure will bedescribed in detail with reference to the appended drawings. Note that,in this specification and the appended drawings, structural elementsthat have substantially the same function and structure are denoted withthe same reference numerals, and repeated explanation of thesestructural elements is omitted. The description will be made in thefollowing order.

1. Microparticle sorting apparatus and microchip for microparticlesorting that are capable of implementing the microparticle sortingmethod according to an embodiment of the present technology

(Overall configuration of the apparatus)

(Microchip configuration)

2. Microparticle sorting method according to an embodiment of thepresent technology

(Sorting operation)

(Counter Flow)

(Drive signal)

3. Modified example of the microparticle sorting method according to anembodiment of the present technology

4. Microparticle sorting program

1. Microparticle Sorting Apparatus and Microchip for MicroparticleSorting that are Capable of Implementing the Microparticle SortingMethod According to an Embodiment of the Present Technology

(Overall Configuration of the Apparatus)

FIG. 1 is a diagram illustrating a configuration of a microparticlesorting apparatus A that is suited to implementing the microparticlesorting method according to an embodiment of the present technology.Further, FIGS. 2 to 4 are diagrams illustrating a configuration of amicrochip 1 a that is mounted on the microparticle sorting apparatus A.FIG. 2 is a top view, FIG. 3 is a perspective view, and FIG. 4 is across-sectional view along the cross-section Q-Q in FIG. 2.

The microparticle sorting apparatus A includes a microchip 1 a, anirradiation unit 21, a detection unit 22, and a drive unit 23. On themicrochip 1 a is formed a main channel 15 through which a fluid (samplefluid) including microparticles that are the target of analysis (referto FIG. 2). Further, an actuator 31 is arranged on the surface of themicrochip 1 a (refer to FIG. 3).

The irradiation unit 21 irradiates light (excitation light) on themicroparticles flowing through the main channel 15 on the microchip 1 a.The irradiation unit 21 includes, for example, a light source that emitsexcitation light and an objective lens that focuses the excitation lighton the microparticles flowing through the main channel 15. The lightsource may be appropriately selected based on the analysis objectivefrom among a laser diode, a SHG laser, a solid laser, a gas laser, ahigh luminance LED and the like. The irradiation unit 21 can optionallyalso have optical elements other than the light source and the objectivelens.

The detection unit 22 detects fluorescence and scattered light that areemitted from the microparticles due to the irradiation with excitationlight. The detection unit 22 includes an objective lens, which focusesthe fluorescence and scattered light emitted from the microparticles, adetector and the like. The detection unit 22 may optionally also haveoptical elements other than the objective lens and the detector.

The fluorescence that is detected by the detection unit 22 may befluorescence emitted from the microparticles themselves or fluorescenceemitted from a fluorescent substance that is labeled on themicroparticles. Further, the scattered light that is detected by thedetection unit 22 may be various types of scattered light, such asforward scattered light, side scattered light, Rayleigh scattered light,and Mie scattering.

The fluorescence and scattered light detected by the detection unit 22are converted into an electric signal, and the electric signal is outputto the drive unit 23. The drive unit 23 determines the opticalcharacteristics of the microparticles based on the input electricsignal. Further, the drive unit 23 has a function for collectingmicroparticles that have been determined to satisfy a predeterminedcharacteristic from the main channel 15 in a sorting channel 16 byapplying a voltage to the actuator 31 and controlling that voltage. Thisfunction of the drive unit 23 will be described in more detail below.The drive unit 23 is configured from a hard disk in which programs andan OS for executing the below-described various processes are stored, aCPU, a memory and the like.

(Microchip Configuration)

The configuration of the microchip 1 a will now be described in moredetail with reference to FIGS. 2 to 4. A sample fluid that includesmicroparticles is introduced from a sample fluid inlet 11 into a samplefluid channel 12. Further, a sheath fluid is introduced from a sheathfluid inlet 13. The sheath fluid introduced from the sheath fluid inlet13 is split and fed into two sheath fluid channels 14 and 14. The samplefluid channel 12 and the sheath fluid channels 14 and 14 merge to formthe main channel 15. A sample fluid laminar flow fed through the samplefluid channel 12 and a sheath fluid laminar flow fed through the sheathfluid channels 14 and 14 merge in the main channel 15, and form a sheathflow in which the sample fluid laminar flow is sandwiched by the sheathfluid laminar flow.

Further, the sheath fluid introduced from the sheath fluid inlet 13 isalso fed to a sheath fluid bypass channel 18 that is formed separatelyto the sheath channel 14. One end of the sheath fluid bypass channel 18is connected to the sheath fluid inlet 13, and the other end isconnected in the vicinity of the communication opening to the mainchannel 15 of a below-described sorting channel 16 (refer to FIG. 4).Although the sheath fluid introduction end of the sheath fluid bypasschannel 18 may be connected to any site where the sheath fluid isflowing, including the sheath fluid inlet 13 and the sheath fluidchannels 14 and 14, it is preferred that the sheath fluid bypass channel18 is connected to the sheath fluid inlet 13. By connecting the sheathfluid bypass channel 18 at a center position (i.e., in the presentembodiment, at the sheath fluid inlet 13) where the two sheath fluidchannels 14 are geometrically symmetrical, equal amounts of the sheathfluid flow can be made to flow to the two sheath fluid channels 14.Reference numeral 156 in FIG. 4 denotes a communication opening of thesorting channel 16 to the main channel 15, and reference numeral 181denotes a discharge opening to the sorting channel 16 of the sheathfluid that is fed through the sheath fluid bypass channel 18.

Reference numeral 15 a in FIG. 2 denotes a detection area whereexcitation light is irradiated by the irradiation unit 21 andfluorescence and scattered light are detected by the detection unit 22.The microparticles are fed to the detection area 15 a in a single linearranged in the sheath flow formed in the main channel 15, and areirradiated with the excitation light from the irradiation unit 21.

The main channel 15 splits into three channels downstream from of thedetection area 15 a. A configuration of the branching portion of themain channel 15 is illustrated in FIGS. 5A, 5B and 5C. Downstream fromthe detection area 15 a, the main channel 15 is in communication withthree branch channels, the sorting channel 16 and waste channels 17 and17. Of these, the sorting channel 16 is a channel into whichmicroparticles that have been determined by the drive unit 23 to satisfya predetermined optical characteristic (hereinafter referred to as“target particles”) are collected. On the other hand, microparticlesthat are determined by the drive unit 23 as not satisfying thepredetermined optical characteristic (hereinafter referred to as“non-target particles”) are not collected in the sorting channel 16, andflow into either of the two waste channels 17 and 17.

The sheath fluid bypass channel 18 is connected to the discharge opening181 positioned near the communication opening 156 to the main channel 15of the sorting channel 16 (refer to FIG. 4). The sheath fluid introducedfrom the sheath fluid inlet 13 is introduced from the discharge opening181 into the sorting channel 16, and forms a sheath fluid flow at thecommunication opening 156 that flows from the sorting channel 16 sidetoward the main channel 15 side (this flow will be described in moredetail blow).

The microchip 1 a is formed from three substrate layers. The samplefluid channel 12, the sheath flow channel 14, the main channel 15, thesorting channel 16, and the waste channel 17 are formed by a firstsubstrate layer a1 and a second substrate layer a2 (refer to FIG. 4). Onthe other hand, the sheath fluid bypass channel 18 is formed by thesecond substrate layer a2 and a third substrate layer a3. The sheathfluid bypass channel 18 formed by the substrate layers a2 and a3 isconnected with the sheath fluid inlet 13 and the discharge opening 181of the sorting channel 16 without being in communication with the samplefluid channel 12, the sheath channel 14, or the main channel 15. Theconfiguration of the sheath fluid inlet 13 side end and of the dischargeopening 181 side end of the sheath fluid bypass channel 18 isillustrated in FIGS. 6 and 7, respectively.

It is noted that the layer configuration of the substrate layers of themicrochip 1 a is not limited to three layers. Further, the configurationof the sheath fluid bypass channel 18 is also not limited to thatillustrated in the drawings, as long as the sheath fluid bypass channel18 is connected with the sheath fluid inlet 13 and the discharge opening181 of the sorting channel 16 without meeting the sample fluid channel12, the sheath channel 14, or the main channel 15.

The collecting of the target particles into the sorting channel 16 isperformed by generating a negative pressure in the sorting channel 16with the actuator 31 to suck the sample fluid including the targetparticles and the sheath fluid into the sorting channel 16. The actuator31 is a piezo element or similar device. The actuator 31 is arranged incontact with the surface of the microchip 1 a, at a positioncorresponding to the sorting channel 16. More specifically, the actuator31 is arranged at a position corresponding to a pressure chamber 161that is provided in the sorting channel 16 as an area whose inner spacehas expanded (refer to FIGS. 3 and 4). The pressure chamber 161 ispositioned downstream from of the communication opening 156 and thedischarge opening 181 in the sorting channel 16.

The inner space of the pressure chamber 161 is, as illustrated in FIG.2, expanded in a planar direction (width direction of the sortingchannel 16), and as illustrated in FIG. 4, expanded in a cross-sectionaldirection (height direction of the sorting channel 16). Namely, thesorting channel 16 is expanded in the width direction and in the heightdirection at the pressure chamber 161. In other words, the sortingchannel 16 is formed so that its vertical cross-section increases insize in the flow direction of the sample fluid and the sheath fluid atthe pressure chamber 161.

The actuator 31 causes the pressure in the sorting channel 16 to changevia the surface (contact face) of the microchip 1 a by producing astretching force due to a change in the applied voltage. When a flow isproduced in the sorting channel 16 due to a change in the pressure inthe sorting channel 16, the volume of the sorting channel 16simultaneously changes too. The volume of the sorting channel 16 changesuntil it reaches a volume that is stipulated based on the displacementof the actuator 31 corresponding to the applied voltage. Morespecifically, when a voltage has been applied and the sorting channel 16is in a stretched state, the actuator 31 keeps the volume of thepressure chamber 161 small by pressing against a displacement plate 311forming the pressure chamber 161 (refer to FIG. 4). When the appliedvoltage decreases, the actuator 31 generates a force in a contractingdirection, whereby the pressing against the displacement plate 311weakens and a negative pressure is generated in the pressure chamber161.

In order to efficiently transmit the stretching force of the actuator 31into the pressure chamber 161, as illustrated in FIG. 4, it is preferredto form a recess on the surface of the microchip 1 a at the positioncorresponding to the pressure chamber 161, and arrange the actuator 31in this recessed portion. Consequently, the displacement plate 311 thatserves as the contact face of the actuator 31 can be made thinner, sothat the displacement plate 311 can be easily displaced by changes inthe pressing force generated by expansion and contraction of theactuator 31, allowing the volume of the pressure chamber 161 to change.

In FIGS. 4, 5A, 5B and 5C, reference numeral 156 denotes a communicationopening of the sorting channel 16 to the main channel 15. The targetparticles being fed in the sheath flow formed in the main channel 15 arecollected in the sorting channel 16 from the communication opening 156.To facilitate the collection of the target particles in the sortingchannel 16 from the main channel 15, as illustrated in FIG. 5C, it isdesirable to form the communication opening 156 so as to open onto aposition corresponding to a sample fluid laminar flow S in the sheathflow formed in the main channel 15. The shape of the communicationopening 156 is not especially limited, and may be, for example, a flatopening shape like that illustrated in FIG. 5A, or a notched openingshape like that illustrated in FIG. 5B formed by cutting the channelwalls of the two waste channels 17.

The microchip 1 a can be configured by laminating a substrate layer onwhich the main channel 15 and the like are formed. The formation of themain channel 15 and the like on the substrate layer can be carried outby injection molding of a thermoplastic resin using a mold. Examples ofthermoplastic resins that can be used include plastics that are known asrelated-art microchip materials, such as polycarbonate, polymethylmethacrylate resin (PMMA), cyclic polyolefins, polyethylene,polystyrene, polypropylene, and polydimethylsiloxane, (PDMS).

2. Microparticle Sorting Method According to an Embodiment of thePresent Technology

(Sorting Operation)

Next, the operation of the microparticle sorting apparatus A will bedescribed.

When the user starts analysis, the microparticle sorting apparatus Adrives a pump to feed the sample fluid and the sheath fluid to thesample fluid inlet 11 and the sheath fluid inlet 13 of the microchip 1a. Consequently, a sheath flow in which the sample fluid laminar flow issandwiched by the sheath fluid laminar flow is formed in the mainchannel 15.

The microparticles are fed to the detection area 15 a in a single linearranged in the sheath flow, and are irradiated by the excitation lightfrom the irradiation unit 21. Fluorescence and scattered light emittedfrom the microparticles due to the irradiation of excitation light aredetected by the detection unit 22, and converted into an electricsignal. The electric signal is output to the drive unit 23.

The drive unit 23 determines the optical characteristics of themicroparticles based on the input electric signal. If a microparticle isdetermined to be a target particle, as illustrated in FIGS. 8A and 8B,after the time (delay period) that the target particle takes to movefrom the detection area 15 a to the branching portion has elapsed, thedrive unit 23 issues a drive signal to the actuator 31 for acquiringthis microparticle. At this point, if necessary, the drive unit 23 canalso be configured to drive the actuator 31 via an amplifier.

Specifically, if the actuator 31 is a piezo element, the drive unit 23produces a negative pressure in the sorting channel 16 by applying avoltage that causes piezo contraction, which causes the volume of thepressure chamber 161 to increase, whereby the target particles collectin the sorting channel 16 from the main channel 15.

On the other hand, if it is determined that a microparticle is not atarget particle, as illustrated in FIGS. 8C and 8D, the drive unit 23issues a non-acquisition drive signal to the actuator 31, and performsoptical characteristics determination of the next microparticle. It isnoted that if the actuator 31 has received a non-acquisition drivesignal, the actuator 31 does not operate.

The drive unit 23 repeats the optical characteristics determination ofthe microparticles and the output of a drive signal to the actuator 31until analysis is finished (refer to FIGS. 8E and 8F), so that only thetarget particles accumulate in the sorting channel 16 (refer to FIG.8F). After analysis has finished, the target particles that have beenseparated into the sorting channel 16 are recovered by the user.

As illustrated in FIG. 9A, the target particles drawn into the sortingchannel 16 are collected in the pressure chamber 161. In the drawing,reference symbol P represents a target particle that has been collectedin the pressure chamber 161, and reference numeral 162 denotes acollection opening for the target particle P into the pressure chamber161. The flow of the sample fluid including the target particle P andthe sheath fluid turns into a jet when flowing into the pressure chamber161, whose interior air has been expanded, and breaks away from thechannel wall face (refer to the arrow in FIG. 9A). Consequently, thetarget particle P separates from the collection opening 162, and iscollected at the back of the pressure chamber 161.

Since the target particles are drawn from the main channel 15 into thepressure chamber 161, the amount of expansion in the volume of thepressure chamber 161 is preferably greater than the volume of thesorting channel 16 from the communication opening 156 until thecollection opening 162 (refer to FIG. 4). Further, the amount ofexpansion in the volume of the pressure chamber 161 is preferably set tobe an amount that generates a negative pressure that is sufficient tocause the flow of the sample fluid including the target particle P andthe sheath fluid to break away from the channel wall face at thecollection opening 162. The drive unit 23 outputs to the actuator 31 apiezo contraction signal with a voltage width that corresponds to thisamount of increase in volume.

Like in the modified example illustrated in FIG. 10, the length of thesorting channel 16 from the communication opening 156 to the collectionopening 162 may be designed to be shorter. The shorter the length fromthe communication opening 156 to the collection opening 162 is, thesmaller the volume of the sorting channel 16 from the communicationopening 156 to the collection opening 16 is. This means that the amountof increase in the volume of the pressure chamber 161 for drawing thetarget particles from the main channel 15 into the pressure chamber 161is smaller. Consequently, the width of the voltage applied on theactuator 31 can be decreased, thereby enabling an efficient sortingoperation.

Thus, by collecting the target particle P at the back of the pressurechamber 161 whose inner space has been expanded in the sorting channel16, the target particle P can be prevented from flowing back out fromthe pressure chamber 161 toward the main channel 15 side even when thepressure in the sorting channel 16 reverses and becomes positive. Asillustrated in FIG. 9B, even when the pressure in the sorting channel 16is positive, since the sample fluid and the sheath fluid flow out over awide area from the vicinity of the collection opening 162, the movementamount of the target particle P itself that has been collected at aposition away from the collection opening 162 is small. Consequently,the target particle P does not flow back out, and is held in thepressure chamber 161.

(Counter Flow)

When the drive unit 23 determines that a microparticle is a non-targetparticle (when a sorting operation is not being performed), it ispreferred that the non-target particle, or a sample fluid containing thenon-target particle, and the sheath fluid do not enter the sortingchannel 16. However, as illustrated in FIG. 11, since the flow of thesample fluid and the sheath fluid fed through the main channel 15 (referto the solid-line arrow in the drawing) has a large momentum, the flowof the sample fluid and the sheath fluid that has flowed from thecommunication opening 156 into the sorting channel 16 changes directionin the sorting channel 16, and flows along the channel wall of thesorting channel 16 and out the main channel 15 side (refer to thedotted-line arrow in the drawing).

The flow of the sample fluid and the sheath fluid that has flowed alongthe channel wall from the sorting channel 16 and out the main channel 15side is slow due to being constricted by the channel wall, so that anaccumulation of the non-target particles, or a sample fluid containingnon-target particles, and the sheath fluid is produced at thecommunication opening 156. This accumulation hinders the sortingoperation of the target particles and the non-target particles frombeing carried out quickly.

In the microparticle sorting apparatus A, the sheath fluid introducedinto the sorting channel 16 from the discharge opening 181 by the sheathfluid bypass channel 18 acts to suppress the non-target particles, or asample fluid containing non-target particles, and the sheath fluid fromentering the sorting channel 16 when a sorting operation is not beingperformed. Namely, the sheath fluid introduced from the sheath fluidinlet 13 is introduced into the sorting channel 16 from the dischargeopening 181, and forms a sheath fluid flow (hereinafter, “counter flow”)at the communication opening 156 that flows from the sorting channel 16side to the main channel 15 side (refer to FIG. 12A). Further, thiscounter flow opposes the flow of the sample fluid and the sheath fluidthat is trying to enter the sorting channel 16 from the main channel 15,thereby inhibiting entry of the sample fluid and the sheath fluid intothe sorting channel 16.

It is preferred that the counter flow has a momentum that matches themomentum (force) of the flow of the sample fluid and the sheath fluidthat is trying to enter the sorting channel 16 from the main channel 15.The momentum of the counter flow can be controlled by adjusting theamount of sheath fluid that is fed to the sheath fluid bypass channel18. This fed amount can be controlled by adjusting the channel diameterof the sheath fluid bypass channel 18. Further, the adjustment of thefed amount can also be carried out using a feed unit such as a syringepump or a valve provided in the sheath fluid bypass channel 18.

The flow rate ratio of the flow rate of the sheath fluid introduced fromthe sheath fluid inlet 13 to the sheath channel 14 to the flow rate tothe sheath fluid bypass channel 18 is determined based on the flowresistance ratio of both channels. Consequently, even if theintroduction pressure of the sheath fluid to the sheath fluid inlet 13varies, a stable operation can be carried out without this flow rateratio fluctuating. Further, even if the sheath fluid flow rate ischanged in order to change the flow velocity of the microparticles atthe detection area 15 a, the flow rate to the sheath channel 14 and theflow rate to the sheath fluid bypass channel 18 may be individuallycontrolled.

It is preferred that the momentum of the counter flow is set so that itis large enough to completely suppress the entry of the sample fluid andthe sheath fluid into the sorting channel 16 from the main channel 15.However, it is acceptable if the counter flow does not completelysuppress such entry. As long as the counter flow reduces entry to someextent, the counter flow can contribute to an increase in the speed ofthe sorting operation. As described above, when the flow of the samplefluid and the sheath fluid that flows along the channel wall from thesorting channel 16 and out the main channel 15 side is produced, thiscauses the non-target particles, or the sample fluid containingnon-target particles, to accumulate. As illustrated in FIG. 12B, if theentry of the sample fluid and the sheath fluid into the sorting channel16 from the main channel 15 can be reduced by a certain extent, the flowof the sample fluid and the sheath fluid that flows along the channelwall from the sorting channel 16 and out the main channel 15 side whichcauses accumulation can be suppressed.

It is noted that by suppressing the accumulation at the communicationopening 156 of the non-target particles, or a sample fluid containingnon-target particles, and the sheath fluid, the target particles and thenon-target particles can be prevented from adhering to the channelwalls.

The counter flow is formed at the communication opening 156 even whenthe target particles are being drawn into the sorting channel 16 (duringthe sorting operation) (refer to FIG. 13A). Consequently, during thesorting operation, the target particles are drawn into the sortingchannel 16 at a drawing pressure that is greater than the counter flow(refer to FIG. 13B). The amount of increase in the volume of thepressure chamber 161 is set so as to be sufficient to generate a drawingpressure greater than the counter flow. The drive unit 23 outputs to theactuator 31 a piezo contraction signal having a voltage width thatcorresponds to this amount of increase in the volume.

In addition, as illustrated in FIG. 13B, the target particles are drawninto the sorting channel 16 until a position that is past the dischargeopening 181. If the drawing into the sorting channel 16 is insufficient,the target particles may flow back out to the main channel 15 due to thecounter flow that is formed by the sheath fluid introduced into thesorting channel 16 from the discharge opening 181 by the sheath fluidbypass channel 18.

To sufficiently draw the target particles until a position that isbeyond the discharge opening 181, the amount of increase in the volumeof the pressure chamber 161 is set to be greater than the flow rate ofthe counter flow, and the flow rate of the flow of the sample fluid andthe sheath fluid that is sucked into the sorting channel 16 from themain channel 15 due to negative pressure is also set to be greater thanthe flow rate of the counter flow. The drive unit 23 outputs to theactuator 31 a piezo contraction signal having a voltage width thatcorresponds to this amount of increase in the volume.

(Drive Signal)

The waveform of the voltage (drive signal when acquiring the targetparticles) applied on the actuator 31 from the drive unit 23 will now bedescribed with reference to FIGS. 14A, 14B and 14C. The waveform of thevoltage applied on the actuator 31 may be any of a “pulse waveform”(FIG. 14A), a “step waveform” (FIG. 14B), or an “undershoot-stepwaveform” (FIG. 14C).

Here, “undershoot-step waveform” means a waveform obtained by adding toa “step waveform” an undershoot portion in which the voltage value islower than the step portion. The “undershoot-step waveform” can be saidto be a combined wave of the “step waveform” and the “pulse waveform”.

The decrease width in the voltage value of the step waveform and thewaveform portion in the undershoot-step waveform is set so as to give asufficient increase in volume to the pressure chamber 161 in order togenerate in the sorting channel 16 a drawing pressure that exceeds thecounter flow at the communication opening 156. Further, this decreasewidth is set so as to cause a sufficient increase in the volume of thepressure chamber 161 in order to draw the target particles into thesorting channel 16 until a position that is past the discharge opening181 due to a negative pressure.

It is preferred that the voltage applied on the actuator 31 is anundershoot-step waveform. With an undershoot-step waveform, the volumeof the pressure chamber 161 can be increased immediately after thesignal starts to be generated, and a large negative voltage can begenerated in the sorting channel 16. Consequently, with anundershoot-step waveform, immediately after starting to draw in thetarget particles, a response to the increase in the collection volume ofthe sample fluid and the sheath fluid inlet in the sorting channel 16from the main channel 15 can be made more quickly, which enables thetarget particles to be collected more rapidly.

In addition to the conditions for satisfying the step waveform and theundershoot-step waveform, the amplitude of the pulse waveform is set soas to give a sufficient increase in volume to the pressure chamber 161in order to draw the target particles from the main channel 15 into thepressure chamber 161 and to make the flow of the sample fluid includingthe target particles and the sheath fluid break away from the channelwall face at the collection opening 162.

Since the pulse waveform and the undershoot-step waveform include awaveform component that causes piezo expansion, the volume of thepressure chamber 161 increases, so that a positive pressure is generatedin the sorting channel 16. Further, in the step waveform too, a positivepressure can be generated in the sorting channel 16 due to unexpectedfluctuations in the voltage value. As described above, since the targetparticle P is collected at the back of the pressure chamber 161, thetarget particle P does not flow back out from the pressure chamber 161toward the main channel 15 side even if a positive pressure is producedin the sorting channel 16.

As the waveform of the voltage applied on the actuator 31, it isparticularly preferred to employ a pulse waveform. For the step waveformand undershoot-step waveform, if the voltage applied on the actuator 31is zero, the actuator 31 reaches the limit of its movable range, andtarget particles are incapable of being collected. This means that thereis a limit to the maximum number of sortable microparticles. On theother hand, for the pulse waveform, there is no such limit.

Thus, according to the microparticle sorting method according to anembodiment of the present technology, improper entry of the sample fluidand the sheath fluid into the sorting channel 16 from the main channel15 can be suppressed due to the formation of a counter flow at thecommunication opening 156 between the main channel 15 and the sortingchannel 16. Consequently, in the microparticle sorting method accordingto an embodiment of the present technology, accumulation of thenon-target particles, or a sample fluid containing non-target particles,and the sheath fluid can be prevented, and the sorting operation of thetarget particles and the non-target particles can be rapidly carriedout.

3. Modified Example of the Microparticle Sorting Method According to anEmbodiment of the Present Technology

In the above-described example, a case was described in which the sheathfluid introduced into the sorting channel 16 from the discharge opening181 by the sheath fluid bypass channel 18 forms only a counter flow thatflows toward the main channel 15 side. In this case, a sorting channelend 19 (refer to FIG. 2) may be a closed end.

On the other hand, the sorting channel end 19 may also be an open end(refer to FIG. 15). In this case, the sheath fluid introduced into thesorting channel 16 from the discharge opening 181 can be split into acounter flow that flows toward the main channel 15 side and a flow(hereinafter referred to as “forward flow”) that flows toward thesorting channel end 19 side.

When control of the voltage applied on the actuator 31 from the driveunit 23 unexpectedly fluctuates, a voltage that causes piezo expansionis applied on the actuator 31, which can produce a positive pressure inthe sorting channel 16. Further, a positive pressure can also beproduced in the sorting channel 16 when a pressure fluctuation in themain channel 15 and the waste channel 17 occur (especially, a decreasein the back-pressure of the waste channel 17). If such a positivepressure is produced, the target particles that have been collected inthe sorting channel 16 may flow back out into the main channel 15.

Due to the formation of the above-described forward flow, the targetparticles that have been drawn into the sorting channel 16 until aposition past the discharge opening 181 are fed further to the back ofthe sorting channel 16 by the forward flow. Consequently, even if thepressure in the sorting channel 16 is positive, the target particles canbe held in the sorting channel 16 without flowing against the forwardflow and back out to the main channel 15. Therefore, the control of thedrive voltage to the actuator 31 can be carried out under robustconditions.

If the sorting channel end 19 is an open end, the sample fluid includingthe target particles and the sheath fluid discharged from the sortingchannel end 19 are recovered in a container via a tube or the likeconnected to the sorting channel end 19. To suppress dilution of therecovered target particles, it is preferred that the flow rate of theforward flow is lower than the flow rate of the counter flow. The flowrate ratio between the forward flow and the counter flow can be adjustedby appropriately changing the channel diameter of the sorting channel16. It is noted that the non-target particles that have flowed to thewaste channel 17 may be accumulated in the waste channel 17 or beexternally discharged. The waste channels 17 and 17 can also bere-merged so as to configure a single external discharge opening for thenon-target particles.

Further, in the above-described example, a case was described in which acounter flow is formed by feeding the sheath fluid introduced from thesheath fluid inlet 13 into the sorting channel 16 by the sheath fluidbypass channel 18. In this case, the counter flow can be formed by asimple chip structure. However, in the microparticle sorting methodaccording to an embodiment of the present technology, as long as acounter flow can be formed at the communication opening 156 between mainchannel 15 and the sorting channel 16, the fluid for forming the counterflow is not limited to the sheath fluid. In addition, the method forfeeding the fluid into the sorting channel 16 is also not limited toemploying the sheath fluid bypass channel 18. For example, a feed unitsuch as a syringe pump may be directly connected to the dischargeopening 181.

4. Microparticle Sorting Program

A microparticle sorting program for executing the above-describedoperations is stored in the drive unit 23 of the above-describedmicroparticle sorting apparatus A.

The program is stored on a hard disk, read into a memory under thecontrol of the CPU and OS, and executes the above-described sortingoperation. The program can be recorded on a computer-readable recordingmedium. The recording medium may be any recording medium as long as itis a computer-readable recording medium. Specifically, a disk-shapedrecording medium may be used, such as a flexible disk and a CM-ROM.Further, a tape type recording medium may be used, such as a magnetictape. In addition, a configuration can also be employed in which a partof the processing may be configured from hardware, such as a DSP(digital signal processor), an ASIC (application specific integratedcircuit), a PLD (programing logic device), and a FPGA(field-programmable gate array), and high-speed processing is performedin cooperation with the above-described software program.

It should be understood by those skilled in the art that variousmodifications, combinations, sub-combinations and alterations may occurdepending on design requirements and other factors insofar as they arewithin the scope of the appended claims or the equivalents thereof.

Additionally, the microparticle sorting method according to theembodiment of the present technology may also be configured as below.

(1) A microparticle sorting method including:

a procedure of collecting a microparticle in a fluid that flows througha main channel in a branch channel that is in communication with themain channel by generating a negative pressure in the branch channel,

wherein, in the procedure, a flow of a fluid is formed that flows towarda side of the main channel from a side of the branch channel at acommunication opening between the main channel and the branch channel.

(2) The microparticle sorting method according to (1), wherein, in theprocedure, the flow is formed by introducing the fluid into the branchchannel from an introduction opening positioned near the communicationopening in the branch channel.

(3) The microparticle sorting method according to (2), wherein, in theprocedure, a flow rate of the fluid that is sucked into the branchchannel from the main channel due to negative pressure is greater than aflow rate of the fluid introduced into the branch channel from theintroduction opening and fed toward the communication opening.

(4) The microparticle sorting method according to (2) or (3), wherein,in the procedure, the microparticle in the main channel is collectedfrom the communication opening to a position that is past theintroduction opening of the branch channel.

(5) The microparticle sorting method according to any one of (1) to (4),wherein the flow is maintained before and after the procedure.

(6) The microparticle sorting method according to any one of (2) to (5),wherein, in the procedure, the fluid introduced into the branch channelfrom the introduction opening is split into a counter flow that flowstoward the communication opening and a forward flow that flows in anopposite direction.

(7) The microparticle sorting method according to any one of (1) to (6),wherein, in the procedure, the negative pressure is generated by anactuator applying a force that deforms an inner space of the branchchannel to cause a volume of the inner space to increase.

(8) The microparticle sorting method according to any one of (1) to (7),wherein, in the procedure, a change in the negative pressure having apulse waveform, a step waveform, or an undershoot-step waveform isproduced in the branch channel. Additionally, the microchip for sortingmicroparticles according to the embodiment of the present technology mayalso be configured as below.

(9) A microchip for sorting microparticles, including:

a sample fluid introduction opening into which a sample fluid includinga microparticle is introduced;

a sample fluid channel through which the sample fluid introduced fromthe sample fluid introduction opening flows;

a sheath fluid introduction opening into which a sheath fluid isintroduced;

a first sheath fluid channel through which the sheath fluid introducedfrom the sheath fluid introduction opening flows;

a main channel where the sample fluid channel and the first sheath fluidchannel merge;

a branch channel that is in communication with the main channel; and

a second sheath fluid channel that connects the sheath fluidintroduction opening and a sheath fluid discharge opening that ispositioned near a communication opening to the main channel in thebranch channel, and that feeds the sheath fluid introduced from thesheath fluid introduction opening into the branch channel from thesheath fluid discharge opening.

(10) The microchip for sorting microparticles according to (9), whereinthe second sheath fluid channel is not in communication with the samplefluid channel, the first sheath fluid channel, or the main channel.

(11) The microchip for sorting microparticles according to (9) or (10),wherein an actuator for applying a displacement on a contact surface isarranged in contact with a position corresponding to the branch channelon a surface.

(12) The microchip for sorting microparticles according to (11), whereina pressure chamber for producing a change in volume due to thedisplacement is configured in the branch channel.

(13) The microchip for sorting microparticles according to (12), whereinthe communication opening, the sheath fluid discharge opening, and thepressure chamber are arranged in the branch channel in order of mention.

(14) The microchip for sorting microparticles according to any one of(9) to (13), further including:

the two first sheath fluid channels,

wherein the sheath fluid introduction opening is provided at asymmetrical center of the two first sheath fluid channels.

(15) The microchip for sorting microparticles according to any one of(9) to (14), wherein an end on an opposite side to the communicationopening of the branch channel is an open end.

The present disclosure contains subject matter related to that disclosedin Japanese Priority Patent Application JP 2012-180317 filed in theJapan Patent Office on Aug. 16, 2012, the entire content of which ishereby incorporated by reference.

What is claimed is:
 1. A microchip, comprising: a first fluid channelthrough which a first fluid flows in a first direction, wherein thefirst fluid comprises of a plurality of microparticles; a branch channelin communication with the first fluid channel via a first communicationopening; a switching portion communicated with an actuator to generate apressure to deflect a selected microparticle of the plurality ofmicroparticles into the branch channel; and a second fluid channel incommunication with the branch channel via a second communication openingin the branch channel, wherein a second fluid flows from the secondfluid channel to the second communication opening, and the secondcommunication opening is downstream of the first communication opening.2. The microchip according to claim 1, wherein the switching portion isfurther configured to: generate a force based on a change in a voltageapplied to the actuator; and change the pressure in the branch channelbased on the force.
 3. The microchip according to claim 2, wherein theactuator is configured to apply a displacement on a surface of themicrochip based on the applied voltage, and the actuator is at aposition, corresponding to the branch channel, on the surface of themicrochip.
 4. The microchip according to claim 1, wherein the switchingportion comprising a pressure chamber configured to communicate with thebranch channel, and the pressure chamber is configured to produce achange in volume of the branch channel.
 5. The microchip according toclaim 1, wherein at least of a portion of the second fluid flows, in asecond direction opposite to the first direction, through the secondfluid channel from a side of the branch channel towards a side of thefirst fluid channel.
 6. The microchip according to claim 1, furthercomprising two sheath fluid channels introduce sheath fluid flow intothe first fluid channel to form a laminar flow.
 7. The microchipaccording to claim 6, further comprising a first inlet that communicateswith the first fluid channel, a sheath inlet that communicates with thesheath fluid channels, a second inlet that communicates with the secondfluid channel.
 8. The microchip according to claim 7, wherein the twosheath fluid channels comprise a sheath fluid introduction opening at asymmetrical center of the two sheath fluid channels.
 9. The microchipaccording to claim 1, further comprising a waste fluid channelcommunicate with the first fluid channel via the first communicationopening.
 10. The microchip according to claim 1, wherein the actuator ison a surface of the microchip at a position corresponding to a pressurechamber, and the pressure chamber is in the branch channel.
 11. Themicrochip according to claim 10, wherein the surface of the microchipcomprises a recess portion at the position corresponding to the pressurechamber, and the actuator is in the recess portion.
 12. The microchipaccording to claim 1, wherein the actuator is a piezo element.
 13. Amicroparticle sorting device, comprising an actuator, a microchip,comprising: a first fluid channel through which a first fluid flows in afirst direction, wherein the first fluid comprises a plurality ofmicroparticles; a branch channel in communication with the first fluidchannel via a first communication opening; a switching portioncommunicated with the actuator to generate a pressure to deflect aselected microparticle of the plurality of microparticles into thebranch channel; and a second fluid channel in communication with thebranch channel via a second communication opening in the branch channel,wherein a second fluid flows from the second fluid channel to the secondcommunication opening, and the second communication opening isdownstream of the first communication opening.
 14. The microparticlesorting device according to claim 13, further comprising: a light sourceconfigured to irradiate light into the plurality of microparticles; anda detector configured to detect light emitted from the plurality ofmicroparticles.